Project Summary Most traits that differ between males and females arise from differential expression of shared genes. Despite the ubiquity of sex-biased expression, we still lack an understanding of both the mechanisms that cause sex-specific gene regulation and the process by which male and female expression becomes uncoupled. These questions have fundamental health implications, as sex-biased diseases such as autoimmune disorders and drug responses show significant genotype-sex interactions. My research will establish how sex-specific gene expression originates by studying a female-limited color dimorphism (FCD) in Drosophila serrata, a species where females have two color morphs (light or dark) but males have only one color morph (only light). Identifying the female dimorphic element and how it functions will provide a useful counterpoint to studies of male-limited pigmentation, enabling me to determine if alleles evolve female- and male-specificity by the same rules. Last year, I mapped female pigmentation to a non-coding structural variant (SV) in the first intron of POU domain motif 3 (pdm3), a transcription factor that I showed represses dark pigmentation. At this variant, dark females have an allele that is radically divergent in size and sequence from the light allele, suggesting that the SV harbors a cis-regulatory element that directs female- and morph-specific pigmentation. Using transgenic experiments, I have confirmed that the sequences flanking the SV function as tissue-specific regulatory elements. In this proposal, I will (1) determine how the D. serrata SV affects pdm3 expression in a binary manner, (2) identify if this SV is regulated sex-specifically because it is controlled by doublesex (the main sex determination effector in Drosophila), and (3) test the necessity and sufficiency of this SV in the origin of FCD using genome editing. This in-depth analysis of a female-specific regulatory element in an experimentally tractable Drosophila model will improve our understanding of how sexual differences arise, and provide a framework for research in other organisms including humans. In particular, this work will inform the design of genetic studies aimed at identifying human alleles that contribute to sex-biased diseases.